Type 1 diabetes is a multifactorial disease caused by an interplay of poorly known environmental factors and partially characterized genetic factors. Focal infiltration of the endocrine pancreas by ...mononuclear cells and a strikingly decreased functional beta cell mass constitute the histopathological hallmarks of the disease at diagnosis, but there is a marked interindividual variability in terms of the extent of the lesions. The disease process is triggered long before clinical onset as testified by the appearance of circulating islet cell autoantibodies years before the development of hyperglycemia. After diagnosis the current insulin substitution therapy is unable to completely avoid the occurrence of the chronic complications of hyperglycemia. The design of effective prevention of hyperglycemia in subjects at risk or of a lasting cure in patients intends to eradicate the development of the invalidating chronic complications. Such clinical studies are complicated by the existence of large interindividual differences in the progression of beta cell destruction, both before and after diagnosis. Therefore it is important to first biologically characterize larger representative groups of patients and subjects at increased risk (e.g. first degree relatives), and to follow them up clinically in order to establish objective criteria for the selection of study subjects with a more homogeneous risk of beta cell destruction for participation in clinical prevention studies. In Belgium a national collaborative program--the Belgian Diabetes Registry--has allowed to collect epidemiological, clinical and biological data in more than 3,500 patients and more than 6,000 relatives. The detection of islet cell-specific autoantibodies--if possible complemented with genetic and hormonal markers--facilitates the identification of subjects at high risk of rapid beta cell destruction. For example, in first degree relatives the combined presence of IA-2 antibodies and the HLA-DQ2/DQ8 genotype defines a small group of subjects (< 1%) with 75% risk of diabetes within 5 years. They qualify for participation in intervention studies aiming at beta cell mass preservation. These findings have helped to prepare several prevention studies in Belgium.
The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive ...function tests such as the proinsulin:C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release.
Between-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5-39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20-57 months (interquartile range).
TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96-0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5-7.1 vs 6.7-9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001) with first-phase C-peptide release during clamp (also with second phase release, only available for age 12-39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release.
The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aims/hypothesis
Thymic expression of self-antigens during T-lymphocyte development is believed to be crucial for preventing autoimmunity. It has been suggested that
G6PC2
, the gene encoding ...islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), is differentially spliced between pancreatic beta cells and the thymus. This may contribute to incomplete elimination of IGRP-specific T lymphocytes in the thymus, predisposing individuals to type 1 diabetes. We tested whether specific splice variation in islets vs thymus correlates with loss of tolerance to IGRP in type 1 diabetes.
Methods
Expression of
G6PC2
splice variants was compared among thymus, purified medullary thymic epithelial cells and pancreatic islets by RT-PCR. Differential immunogenicity of IGRP splice variants was tested in patients and healthy individuals for autoantibodies and specific cytotoxic T lymphocytes using radiobinding assays and HLA class I multimers, respectively.
Results
Previously reported
G6PC2
splice variants, including full-length
G6PC2
, were confirmed, albeit that they occurred in both pancreas and thymus, rather than islets alone. Yet, their expression levels were profoundly greater in islets than in thymus. Moreover, three novel
G6PC2
variants were discovered that occur in islets only, leading to protein truncations, frame shifts and neo-sequences prone to immunogenicity. However, autoantibodies to novel or known IGRP splice variants did not differ between patients and healthy individuals, and similar frequencies of IGRP-specific cytotoxic T lymphocytes could be detected in both patients with type 1 diabetes and healthy individuals.
Conclusions/interpretation
We propose that post-transcriptional variation of tissue-specific self-proteins may affect negative thymic selection, although this need not necessarily lead to disease.
The limitations of current rat C-peptide assays led us to develop a time-resolved fluorescence immunoassay for measurements in plasma, incubation media, and tissue/cell extracts. The assay uses 2 ...monoclonal antibodies, binding to different parts of the C-peptide molecule, and allowing, respectively, capture of the peptide and its detection by europium-labeled streptavidin. It is performed on 25-μL samples for a dynamic range from 66pM up to 3900pM C-peptide and displays over 95% recovery of added peptide in the range of 111pM to 2786pM. Its inter- and intra-assay coefficients of variations are, respectively, lower than 7.6% and 4.8%. Cross-reactivities by rat insulin and by human and porcine C-peptide are negligible, and cross-reactivity by mouse C-peptide is 6% ± 2%. The assay has been validated for in vivo and in vitro measurements of C-peptide release and cellular content. Release patterns were similar to those for insulin and occurred in equimolar concentrations for both peptides. The molar C-peptide contents in purified β-cells and isolated islets were similar to the corresponding insulin contents. This was also the case for pancreatic extracts containing protease inhibitors.
When the concentrations of 2 or more substances are measured separately, their molar ratios are subject to the additive imprecisions of the different assays. We hypothesized that the cumulative error ...for concentration ratios of peptides containing a common sequence might be minimized by measuring the peptides simultaneously with a "trefoil-type" immunoassay.
As a model of this approach, we developed a dual-label time-resolved fluorescence immunoassay (TRFIA) to simultaneously measure proinsulin, C-peptide, and the proinsulin-C-peptide ratio (PI/C). A monoclonal antibody captures all C-peptide-containing molecules, and 2 differently labeled antibodies distinguish between proinsulin-like molecules and true C-peptide.
The trefoil-type TRFIA was capable of measuring plasma C-peptide and proinsulin simultaneously without mutual interference at limits of quantification of 48 and 8125 pmol/L, and 2.1 and 197 pmol/L, respectively. Within-laboratory imprecision values for the trefoil-type TRFIA ranged between 8.4% and 12% for the hormone concentrations. Unlike the hormone results obtained with separate assays, imprecision did not increase when PI/C was calculated from trefoil assay results (P < 0.05). Peptide concentrations were highly correlated with results obtained in individual comparison assays (r(2) > or = 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained < or = 25% over a broader C-peptide range than with separate hormone assays (79-7200 pmol/L vs 590-4300 pmol/L C-peptide). Preliminary data indicate little or no interference by heterophile antibodies.
The developed trefoil-type TRFIA is a reliable method for simultaneous measurement of proinsulin, C-peptide, and PI/C and provides proof of principle for the development of other trefoil-type multiple-label immunoassays.
We present a time-resolved fluorescence immunoassay (TR–FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to ...different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25
μl could be analyzed in less than 2
days with a measuring range between 5 and 1250
pg (0.2–50
μg/L or 34.4–8600
pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40
μg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (
P
<
0.0001,
r
2
=
0.99). The TR–FIA method had a similar detection limit (0.16
μg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.
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